Androgens are compounds which stimulate secondary sex characteristics and produce male secondary sex characteristics. The androgen 17 beta-Hydroxyandrost-4-en-3-one, commonly called testosterone, is synthesized in the interstitial (Leydig) cells of the testis in males. The synthesis of testosterone in the Leydig cells during adulthood is mainly controlled by the levels of pituitary luteinising hormone (LH). In females, there are three sources of testosterone biosynthesis. The adrenal glands and the ovaries secrete small quantities of testosterone, and the peripheral metabolism of androstenedione accounts for 50-60% of the daily testosterone production in normal females.
Testosterone exists in two forms in the blood stream: approximately 99% of the testosterone is bound to plasma proteins and the remainder is unbound. At least three serum proteins bind testosterone. Each protein binds testosterone with differing affinities. At physiological concentrations, the hormone is largely bound to a low capacity, high affinity beta-globulin, designated sex hormone binding globulin (SHBG). A smaller fraction is bound to albumin and to cortisol-binding globulin. The structural formula of testosterone and its numbering system is represented below: ##STR2##
Testosterone is metabolized primarily in the liver. Enzymes have been identified in the skin and the reticuloendothelial system which are capable of metabolizing testosterone. Two major metabolic pathways of testosterone have been identified. In the 17-ketonic pathway, the 17 beta-hydroxy group is oxidized to a ketone. This results in the formation of the weak androgen, androstenedione. The pathway forms intermediate metabolites which have little biological activity. The second pathway, the 17-hydroxy route, involves changes initially in the A ring. In this pathway, the 17 beta-hydroxy group is not altered. This is important because the 17 beta-hydroxy group is required for the potency of androgenic steroids and their intermediate metabolites. Therefore, this metabolic pathway produces intermediate metabolites with considerable androgenic activity.
Clinical Utility
Testosterone measurements are useful in the evaluation of hypogonadal states. Common causes of decreased testosterone in males include: hypogonadism, orchidectomy, estrogen therapy, Klinefelter's syndrome, hypopituitarism, testicular feminization and hepatic cirrhosis.
In females, testosterone levels are normally found to be much lower than those encountered in the normal male. Common causes of increased serum testosterone levels in females include polycystic ovaries (Stein-Leventhal syndrome), ovarian tumors, adrenal tumors and adrenal hyperplasia. Virilization in women is associated with the administration of androgens and endogenous overproduction of testosterone.
Current Testosterone Assays
There are several methods available for the quantification of testosterone in serum. The techniques used to estimate the concentration of testosterone in serum/plasma fall into six main categories: (1) gas chromatography/mass spectroscopy (Sabot, J. F., et al., J. of Chromatography, 339:233 (1985); Shinohara, Y., el. al., Biomedical and Environmental Mass Spectrometry, 16:241 (1988); Furuta, T., et al., J. of Chromatography, 525:15 (1990); Fukushima, S., et al., J. of Chromatography, 565:35 (1991); Wudy, S. A., et al., Steroids, 57:319 (1992)); (2) isotope dilution mass spectrometry (Siekmann, L., J. Steroid Biochem., 11:117 (1979); Moneti, G., et al., J. Steroid Biochem., 27(1-3):53 (1987)); (3) thin layer chromatography (Vingler, P., et al., J. of Chromatography, 571:73 (1991)); (4) chemiluminescence (Syropoulos, A. B., et al., Analytica Chimica Acta, 239:195 (1990); Stabler, T. V., et al., Clin. Chem., 37(11):1987 (1991); Van Dyke and Van Dyke, 1991); (5) high-performance liquid chromatography (Suzuki, Y., et al. J. of Chromatogr., 426:33 (1988); Erkoc, F. U., et al., .J Chromatogr. Sci., 27:86 (1989); Ueshiba, H., et al,, Clin. Chem., 37(8):1329 (1991); (6) enzyme-linked immunoassays (Hosada, H., et. al., Chem. Pharm. Bull., 28(10):3035 (1980); Marcus, G. J., et al., Steroids, 46(6):975 (1985); Ali, E., et al., J. Immunol. Methods, 147:173 (1992); Sengupta, J., et al., J. Immunol. Methods, 147:181 (1992); Dhar, T. K., et al., J. Immunol. Methods, 147:167 (1992); Rassasie, M. J., et al., Steroids, 57:288 (1992); Rassasie, M. J., et al., Steroids, 57:112 (1992); Boehringer Mannheim GmbH, 1992); and finally (7) radioimmunoassay (Rao, P. N., et al., J. Steroid Biochem., 9:539 (1978); Hosada, H., et. al., J. Steroid Biochem., 10:513 (1979); Cekan, S. Z., J. Steroid Blochem., 11:1629 (1979); ICN Biomedicals, Inc., "RSL .sup.125 I Testosterone," Package Insert, Revision No. 4, January 1983; Diagnostics Products Corporation, "Coat-A-Count Total Testosterone," Package Insert, V 116, January 1992.).
The development since the 1960's of extremely sensitive and specific radioimmunoassays (RIAs) revolutionized the quantification of steroids. Because of their speed, simplicity and relatively low cost, the RIA approach is often used. This trend has been greatly encouraged by the availability of convenient, reliable commercial kits.
The commercially available testosterone assays are primarily "direct" radioimmunoassays which do not require organic extraction. In the RIAs, a limited amount of specific antibody is reacted with the hormone. .sup.125 I-labeled testosterone competes for a fixed time with testosterone in the patient sample. After separation of the bound from the free hormone, the amount of radioactivity in the bound fraction is quantified and used to construct a standard curve against which the unknown samples are measured. The commercial assays for testosterone vary in the method used to separate the bound and free hormone.
Examples of commercially available RIAs include the Coat-A-Count Total Testosterone Assay from Diagnostic Products Corporation (DPC) and the ICN Biomedicals, Inc. RSL .sup.125 I-Testosterone kit. The DPC kit utilizes a solid-phase separation procedure: (1) testosterone-specific antibody is immobilized to the wall of a polypropylene tube, (2) .sup.125 I testosterone competes with analyte in the patient specimen, (3) the tube is decanted to separate the bound from the free hormone.
The RSL kit utilizes a liquid phase separation procedure: 1) testosterone-specific antibody is not bound to a solid phase, but is free in solution, 2) .sup.125 I-testosterone competes with analyte in the patient specimen, 3) a precipitating antiserum (second antibody) is then added to precipitate the testosterone-specific antibody:hormone and separate the bound from free hormone.
The above-described RIA methods suffer from the following disadvantages: 1) individuals must use radiological protection procedures; 2) radioactive waste must be disposed; 3) the labeled reagents used have short half-lives. For these reasons alternative immunoassay methods have been sought.
In contrast to the RIA methods, recently introduced microparticle enzyme immunoassays (MEIAs) are relatively new test methods being used in the physician's office and hospital settings. In this method, antibody is bound to the latex particles in suspension and the corresponding analyte being evaluated is then specifically bound. The particles are then passed through a glass fiber filter system. The particles adsorb to the glass fiber filter and the unbound analyte is washed through the glass fiber filter. For small molecular weight analytes such as steroids, the amount of analyte is determined by quantifying the number of antibody molecules which have not bound analyte. This is achieved by the addition of a hapten labeled conjugate. A substrate for the enzyme is then added which generates fluorescence rates which are inversely proportional to the amount of analyte in the sample.
Another nonisotopic homogeneous technique that has gained widespread use is fluorescent polarization. This technology was not known for the quantification of testosterone. Fluorescent polarization techniques are based on the principle that a fluorescent labeled compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Therefore, when a fluorescent labeled tracer-antibody complex is excited with linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted. When a "free" tracer compound (i.e., unbound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate and the molecules are more randomly oriented, therefore, the emitted light is depolarized. Thus, fluorescent polarization provides a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay.